The ideal tissue engineering (TE) strategy for ligament regeneration should recapitulate the bonecalcified cartilage -fibrocartilage -soft tissue interface. Aligned electrospun-fibers have been shown to guide the deposition of a highly organized extracellular matrix (ECM) necessary for ligament TE.However, recapitulating the different tissues observed in the bone-ligament interface using such constructs remains a challenge. This study aimed to explore how fiber alignment and growth factor stimulation interact to regulate the chondrogenic and ligamentous differentiation of mesenchymal stem cells (MSCs). Without growth factor stimulation, MSCs on aligned-microfibers showed higher levels of tenomodulin (TNMD) and aggrecan gene expression compared to MSCs on randomlyoriented fibers. MSCs on aligned-microfibers stimulated with transforming growth factor β3 (TGFβ3) formed cellular aggregates and underwent robust chondrogenesis, evidenced by increased type II collagen expression and sulphated glycosaminoglycans (sGAG) synthesis compared to MSCs on randomly-oriented scaffolds. Bone morphogenetic protein 2 (BMP2) and type I collagen gene expression were higher on randomly-oriented scaffolds stimulated with TGFβ3, suggesting this substrate was more supportive of an endochondral phenotype. In the presence of connective tissue growth factor (CTGF), MSCs underwent ligamentous differentiation, with increased TNMD expression on aligned compared to randomly aligned scaffolds. Upon sequential growth factor stimulation, MSCs expressed types I and II collagen and deposited higher overall levels of collagen compared to scaffolds stimulated with either growth factor in isolation. These findings demonstrate that modulating the alignment of microfibrillar scaffolds can be used to promote either an endochondral, chondrogenic, fibro-chondrogenic or ligamentous MSC phenotype upon presentation of appropriate biochemical cues.